Commentary 539

Non-canonical ubiquitin-based signals for

proteasomal degradation
Yelena Kravtsova-Ivantsiv* and Aaron Ciechanover*
Cancer and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, TechnionIsrael Institute of
Technology, Efron Street, Bat Galim, P.O. Box 9649, Haifa 31096, Israel
*Authors for correspondence (yelenaiv@tx.technion.ac.il; c_tzachy@netvision.net.il)

Journal of Cell Science 125, 539548

2012. Published by The Company of Biologists Ltd
doi:10.1242/jcs.093567

Summary
Regulated cellular proteolysis is mediated largely by the ubiquitinproteasome system (UPS). It is a highly specific process that is
time- (e.g. cell cycle), compartment- (e.g. nucleus or endoplasmic reticulum) and substrate quality- (e.g. denatured or misfolded
proteins) dependent, and allows fast adaptation to changing conditions. Degradation by the UPS is carried out through two successive
steps: the substrate is covalently tagged with ubiquitin and subsequently degraded by the 26S proteasome. The accepted canonical
signal for proteasomal recognition is a polyubiquitin chain that is anchored to a lysine residue in the target substrate, and is assembled
through isopeptide bonds involving lysine 48 of ubiquitin. However, several non-canonical ubiquitin-based signals for proteasomal
targeting have also been identified. These include chains anchored to residues other than internal lysine in the substrates, chains
assembled through linking residues other than lysine 48 in ubiquitin, and mixed chains made of both ubiquitin and a ubiquitin-like
protein. Furthermore, some proteins can be degraded following modification by a single ubiquitin (monoubiquitylation) or multiple
single ubiquitins (multiple monoubiquitylation). Finally, some proteins can be proteasomally degraded without prior ubiquitylation (the
Journal of Cell Science

process is also often referred to as ubiquitination). In this Commentary, we describe these recent findings and discuss the possible
physiological roles of these diverse signals. Furthermore, we discuss the possible impact of this signal diversity on drug development.
This article is part of a Minifocus on Ubiquitin. For further reading, please see related articles: Ubiquitin and SUMO in DNA repair at a glance by Helle D. Ulrich (J.
Cell Sci. 125, 249-254). Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control by Annamaria Mocciaro and Michael Rape (J. Cell Sci. 125, 255-
263). The role of ubiquitylation in receptor endocytosis and endosomal sorting by Kaisa Haglund and Ivan Dikic (J. Cell Sci. 125, 265-275). Cellular functions of the
DUBs by Michael J. Clague et al. (J. Cell Sci. 125, 277-286). HECT and RING finger families of E3 ubiquitin ligases at a glance by Meredith B. Metzger et al. (J.
Cell Sci. 125, 531-537). No one can whistle a symphony alone how different ubiquitin linkages cooperate to orchestrate NF-B activity by Anna C. Schmukle and
Henning Walczak (J. Cell Sci. 125, 549-559).
Key words: Ubiquitin, Polyubiquitin chains, Monoubiquitylation, Proteasome, Protein degradation

Introduction
Ubiquitylation [also known as ubiquitination, as coined by the
discoverers of this modification with regard to its connection to
proteolysis (Wilkinson, 2005)] is a three-step enzymatic reaction
that is carried out by several enzymes: the ubiquitin-activating
enzyme (E1), a ubiquitin carrier protein (E2; also known as
ubiquitin-conjugating enzyme, UBC) and a ubiquitin-protein ligase
(E3). An additional component of the ubiquitylation machinery has
been described. This E4 enzyme is involved in elongation of short
ubiquitin chains (Koegl et al., 1999). However, the requirement for
an E4 activity appears to be limited to a small subset of substrates.
Ubiquitylation-dependent proteasomal degradation is involved in
the regulation of numerous cellular processes, including cell cycle
progression, apoptosis, DNA repair, the maintenance of cellular
quality control, autophagy, the regulation of transcription and
receptor-mediated endocytosis (Mayer et al., 2005; Mayer et al.,
2006; Mayer et al., 2008). In general, modification by ubiquitin
serves as a recognition element in trans, whereby different
downstream effectors bind to the ubiquitin-modified protein to
affect its fate and/or function. In the case of proteasomal
degradation, the ubiquitylated protein is recognized by the 26S
proteasome and subsequently degraded (Dikic et al., 2009; Su and
Lau, 2009).
The widely accepted canonical signal for proteasomal degradation
is a polyubiquitin chain that is anchored to the -NH2 group of a
lysine residue(s) in the substrate by an isopeptide bond and is
assembled through the formation of isopeptide bonds between the
C-terminal residue of one ubiquitin moiety (glycine 76) and lysine
48 of the previously conjugated ubiquitin moiety (Chau et al.,
1989). Recent studies have reported, however, that other types of
ubiquitin chains can also be recognized by the proteasome (Figs 1,
2). These include an ester-based linkage that connects ubiquitin to
a threonine or serine residue in the substrate, and a thiolester-based
linkage whereby ubiquitin is bound to a cysteine residue in the
substrate (McDowell et al., 2010; Tait et al., 2007; Vosper et al.,
2009). Ubiquitin can also be conjugated to the -NH2 group of the
N-terminal residue of the substrate. Instead of using lysine 48 for
the linkage, polyubiquitin chains can also be assembled through
one of the six additional lysine residues in the molecule. Such
homogenous chains based on, for example, lysine 63 (Saeki et al.,
2009), or heterogeneous chains in which different ubiquitin
ubiquitin linkages are found, have also been reported to target
proteins for proteasomal degradation. Linear chains, in which the
ubiquitin links are attached to one another head-to-tail, and
heterologous chains, in which the links are made of ubiquitin and a
ubiquitin-like protein, such as small ubiquitin-like modifier (SUMO),
have additionally been shown to target proteins for proteasomal
degradation. Surprisingly, it has been demonstrated that the
proteasome does not necessarily have to recognize a polyubiquitin
chain or tetraubiquitin, which had been described previously as the
minimal proteasomal targeting signal (Thrower et al., 2000). The
proteasome can also recognize proteins that are modified by a
single ubiquitin moiety (monoubiquitylation) or multiple single
moieties (multiple monoubiquitylation). Finally, a few exceptional
 540 Journal of Cell Science 125 (3)

Fig. 1. Different types of ubiquitin

A Polyubiquitylation chains. (A)Polyubiquitylation.
Homogeneous chains, namely chains
based on linkages involving lysines 6,
11, 27, 29, 33, 48 or 63 (1);

O H
heterogeneous chains (2): mixed chains

C N
(1) Homogenous (3) Heterologous (4) Linear
(SUMOylation and -ubiquitylation) based on linkages involving different
K6, K11, K27, K29
lysines (i) and multiply branched (or
K33, K48 or K63
forked) chains in which several
ubiquitin moieties are anchored to
distinct lysine residues in a single
ubiquitin moiety (ii); heterologous

C N
H
chains are made of both ubiquitin and a

ubiquitin-like protein (3); linear chains
in which the ubiquitin moieties are
linked head-to-tail (the C-terminal
residue of the distal moiety is linked to
the N-terminal residue of the proximal
one) (4). (B)Monoubiquitylation
representing modification of a protein
(2) Heterogenous
by: a single ubiquitin (1) or several
single ubiquitins (2).
(i) Mixed (ii) Multiply branched (C)Oligoubiquitylation, namely the
(forked) modification of a protein by short
ubiquitin chains. Inset: the seven
internal lysines of ubiquitin; K, lysine.
Journal of Cell Science

B Monoubiquitylation

(1) Monoubiquitylation (2) Multiple C Oligoubiquitylation

monoubiquitylation

}
Key K K K K KK K
6 11 27 29 33 48 63 Ubiquitin
NH 2 COOH

cases have been reported, where the proteasome can degrade proteins
that have not been modified by ubiquitin at all.
In addition to the different types of ubiquitin modifications
described above, recent evidence supports the idea that there are
signals that consist of multiply branched (forked) chains where
two (or perhaps even more than two) ubiquitin molecules are
linked to a single ubiquitin moiety. In contrast with those chains
that target proteins for proteasomal degradation, these chains
cannot be efficiently processed by the 26S complex (Kim et al.,
2007) and serve non-proteolytic functions (Ben-Saadon et al.,
2006).
The broad diversity of ubiquitin-based signals suggests a high
level of specificity and selectivity in proteasomal recognition and
degradation of proteins. However, our knowledge of the formation

and the biological significance of the variety of these non-canonical
proteasomal signals is scarce. It is still unclear which features
within the substrate and/or E3 ligase are important for the
generation of a specific ubiquitin signal, and whether the existence
of numerous signals represents diversity among the acceptor
proteins that shuttle substrates to the proteasome and within the
proteasome complex itself.
In this Commentary, we describe the evolving repertoire of non-
canonical ubiquitin-based proteasomal signals, and review the
mechanism of proteasomal degradation of substrates that are
degraded in a ubiquitin-independent manner. In particular, we
discuss the possible mechanisms that govern the diversity of signals
for proteasomal targeting. Importantly, aberrations in the ubiquitin
system underlie the pathogenesis of numerous diseases, such as
certain forms of neurodegeneration, inflammatory disorders and
malignancies. Consequently, the system has served as a platform
for novel mechanism-based drug development, including one
successful drug that is already in widespread use to combat multiple
myeloma (Velcade). Therefore, one can envision that the broad
diversity of proteolytic signals will serve as an even broader
platform for the development of specific drugs in the future.
Ubiquitin-dependent degradation
The diversity of the proteasomal substrates along with that of their
Journal of Cell Science
targeting signals underlies the versatility and complexity of the
UPS. Here, we will describe recent findings on the proteasomal
degradation of substrates harboring a variety of non-canonical
ubiquitin signals that do not involve lysine-48-based chains.
Ubiquitylation of substrates on lysine residues
Homogenous polyubiquitin chains
The ubiquitin molecule contains seven lysine residues in positions
6, 11, 27, 29, 33, 48 and 63. Studies in yeast have shown that,
under certain conditions, any of them can be involved in formation
of homogeneous polyubiquitin chains (Peng et al., 2003; Xu et al.,
2009; Ziv et al., 2011). Complementing studies in both yeast and
mammalian cells have shown that the chains based on linking
ubiquitin residues through six out of the seven lysines (namely all
but lysine 63) can target proteins for degradation (Bedford et al.,
2011; Xu et al., 2009). Corroborating these data, a mass
spectrometry analysis has shown that, although the abundance of
the different linkages varies, homogeneous polyubiquitin chains
based on linkages involving lysines 6, 11, 27, 29 and 48 can all
mediate proteasomal degradation (Dammer et al., 2011).
The analysis of ubiquitin chains that target specific substrates
for proteasomal degradation has revealed that the E3 ligase
C-terminus of HSP70-interacting protein (CHIP) synthesizes
polyubiquitin chains that are linked through lysines 6, 11, 48 or 63
on its targets, the molecular chaperones heat-shock proteins HSP70
and HSP90 (Kundrat and Regan, 2010). Studies carried out by
other research groups have confirmed the involvement of lysine-
63-based chains in targeting proteins for proteasomal degradation.
For example, it has been shown in vitro that the modification of
the model substrate ubiquitin-dihydrofolate reductase (DHFR) by
lysine-63-linked tetraubiquitin chains bound to lysine 48 of the
fused ubiquitin moiety, results in its proteasomal degradation
(Hofmann and Pickart, 2001). In a cell-free system, troponin I has
been shown to be targeted for proteasomal degradation by the
muscle-specific ubiquitin RING finger protein 1 (MuRF1, also
known as TRIM63) ligase, which, depending on which E2 is used
(UBCH1, also known as E2-25kDa, or heterodimeric UBCH13
Non-canonical proteasomal signals 541
A Internal lysines B N-terminal residue
O H
C N
C Residues other than
lysine (Cys, Ser, Thr)
Key
}
K K K K KK K
C S 6 11 27 29 33 48 63 Ubiquitin
T
NH 2 COOH
Fig. 2. Different ubiquitylation sites on target substrates. (A)Internal
lysines. An isopeptide bond is generated between the C-terminal glycine 76 of
ubiquitin and an -NH2 group of a lysine residue in the substrate. (B)N-
terminal residue. A linear peptide bond is created between the C-terminal
glycine 76 of ubiquitin and the -NH2 group of the N-terminal residue.
(C)Residues other than lysine (cysteine, serine, threonine). An ester bond is
created between the C-terminal glycine 76 of ubiquitin and a serine or a
threonine residue in the substrate. A thiolester bond is created between the
C-terminal glycine 76 of ubiquitin and a cysteine residue. Cys (or C) denotes
cysteine, Ser (or S) denotes serine, and Thr (or T) denotes threonine.
UEV1A, respectively), synthesizes lysine-48- or lysine-63-based
ubiquitin chains (Kim et al., 2007). The Rsp5 ubiquitin ligase
ubiquitylates the ER membrane-anchored transcription factor Mga2,
thereby generating chains that are highly rich in lysine 63 linkages
(Saeki et al., 2009). These chains target the substrate for
proteasomal processing. Interestingly, lysine-63-linked chains have
been detected in various proteasome-bound polyubiquitylated
proteins, suggesting that they have a contribution to proteasomal
recognition (Saeki et al., 2009). It should be taken into
consideration, however, that most of these experiments were carried
out using purified components in cell-free assays, and the
involvement of such chains in cellular proteasomal degradation is
not clear. Furthermore, it should be noted that in both yeast and
mammalian cells, lysine-63-based chains have been shown to target
mainly membrane proteins for degradation in the vacuole and
lysosome, respectively (Lauwers et al., 2010). It appears that these
chains are highly specific and cannot be replaced by chains with
other linkages. In addition, lysine-63-based chains have important
roles in intracellular signaling where the modifications serve non-
proteolytic purposes (Chen and Sun, 2009).
The anaphase-promoting complex/cyclosome (APC/C) is an E3
ligase that coordinates progression through the cell cycle by
modifying a variety of cell cycle regulators. Several studies have
shown that lysine-11-linked chains are crucial regulators of mitotic
protein degradation (Jin et al., 2008; Kirkpatrick et al., 2006;
Matsumoto et al., 2010), and that this type of ubiquitin modification
is upregulated in mitotic human cells (Matsumoto et al., 2010).
 542 Journal of Cell Science 125 (3)
Among the substrates that are targeted by APC/C and modified by
lysine-11-linked chains are proteins that are required for spindle
assembly, such as BRCA1-associated RING domain protein 1
(BARD1), hyaluronan-mediated motility receptor (HMMR),
hepatoma upregulated protein (HURP, also known as DLGP5),
and nucleolar and spindle-associated protein (NUSAP1) (Song and
Rape, 2010). Two E2s are required for APC/C to assemble these
specific lysine-11-linked chains: the ubiquitin chain initiator
UBCH10 (also known as UBE2C) that primes formation of the
chain, and the chain elongator ubiquitin-conjugating enzyme E2S
(UBE2S) (Garnett et al., 2009; Williamson et al., 2009; Wu et al.,
2010). The formation of these chains depends on the TEK box
motif on the ubiquitin surface. Homologous TEK boxes were also
found on APC/C substrates. It is therefore possible that recognition
of the TEK box on both ubiquitin and the substrate enables APC/C
to efficiently synthesize the lysine-11-based chains (Jin et al.,
2008). The observation that lysine-11-linked chains are able to
compete with lysine 48 chains for binding to the S5 subunit of the
proteasome (Baboshina and Haas, 1996), further strengthens the
notion that the proteasome has the ability to recognize these chains.
In addition to the substrates modified by APC/C, the E2E3 protein
complex comprising the ubiquitin-conjugating enzyme H5a
(UBCH5a) and the ligase seven in absentia homolog 1 (SIAH1)
can assemble lysine-11-linked ubiquitin chains on -catenin and
Journal of Cell Science
thereby cause its proteasomal degradation (Dimitrova et al., 2010).
Taken together, it appears that chains based on all lysine residues
in ubiquitin can target substrates for proteasomal degradation.
However, one should be cautious in drawing such a broad conclusion,
as some of the experiments were carried out in cell-free systems,
using purified recombinant components that might not be in
physiological concentrations, and might therefore not faithfully
represent cellular events. In addition, the involvement of
polyubiquitin chains based on lysines 6, 27, 29 and 33 in the
degradation of specific cellular proteins has not yet been directly
demonstrated, and the conclusion that they are involved in
proteasomal degradation is based mostly on mass spectrometry
analysis of entire cellular proteomes under non-perturbed and
perturbed (i.e. following addition of the proteasome inhibitor MG132)
conditions (Xu et al., 2009). Furthermore, cells contain numerous
ubiquitin ligases, and the chains that they generate are not necessarily
homogenous and might contain more than one type of lysine-based
linkage (Fig. 1). The probable presence of mixed ubiquitin chains
makes it more difficult to evaluate the precise roles of individual
non-canonical homogeneous linkages in targeting substrates for
degradation. As a matter of fact, one can argue that a critical mass
of a certain linkage, for example a segment of four ubiquitins linked
through lysine 48, can be sufficient for targeting the substrate for
degradation, whereas the other linkages contribute very little, or not
at all, to the proteolytic process and were generated spuriously by
the primary or secondary ligases. These arguments suggest the
possibility of non-specific creation of linkages and indicate that
further experiments are required to elucidate the precise relevance of
these protein modifications for proteasomal degradation in vivo.
Overall, accumulating experimental evidence suggests that all
lysine residues in ubiquitin can be involved in the formation of
chains targeting proteins for proteasomal degradation, although the
experimental evidence is stronger for certain linkages and weaker
for others. It is also difficult to state with certainty that the chains
in cells are indeed homogenous. When assuming that different
linkages have a role in cellular proteolysis, it should be noted that
such chains are similar to lysine-48-based chains in the sense that
they are assembled through a single linkage type. Nonetheless,
they are clearly different from one another structurally, and therefore
might be recognized by different shuttle proteins and proteasomal
subunits, or serve to modulate the degradation rate of their target
substrates.
Heterogeneous ubiquitin chains
For a long time, it was assumed that polyubiquitin chains are
homogeneous, and all moieties are attached to each other through
the same internal lysine residue. However, mixed chains, where
different moieties are bound through different lysine residues have
been identified, mostly through mass spectrometry analysis of
cellular and cell-free adducts (e.g. Kirkpatrick et al., 2006).
Moreover, multiply branched (or forked) chains in which two (or
perhaps even more) ubiquitin moieties are anchored to distinct
lysine residues in a single moiety have been described (Ben-Saadon
et al., 2006; Kim et al., 2007). In some cases, it has been shown
that the ubiquitin moieties are linked to neighboring lysine residues,
e.g. lysines 6 and 11, 27 and 29, or 29 and 33 (Kim et al., 2007).
However, it should be taken into account that these results are
based on mass spectrometry, which involves treatment of samples
with trypsin. Therefore, if two ubiquitin moieties were linked to
nonadjacent lysines of the proximal ubiquitin, they will be
separated, and only the neighboring forks will remain intact and
will therefore be detected. In another study, the ubiquitin moieties
were shown to be linked to more distant residues (lysines 6, 27 and
48) (Ben-Saadon et al., 2006). Here, the researchers carried out in
vitro ubiquitylation assays using different ubiquitin species with
point mutations of different internal lysines.
In contrast with branched homogeneous ubiquitin chains, forked
chains (Fig. 1) bind to the proteasome substantially less efficiently,
and substrates tagged with these chains are degraded more slowly
(Kim et al., 2007; Kim et al., 2009). It appears that the function of
forked chains in degradation is negligible. In agreement with this,
forked chains formed on RING1B (also known as RING2), the
ligase component of the polycomb repressive complex 1 (PRC1,
also known as PCGF1), serve a non-proteolytic function and instead
stimulate the monoubiquitylating ligase activity of RING1B
towards its substrate, histone H2A (Ben-Saadon et al., 2006).
Furthermore, APC/C along with UBCH10 monoubiquitylates
multiple lysine residues in cyclin B1. These ubiquitin moieties are
then converted to mixed polyubiquitin chains (Fig. 1) that are
enriched with linkages based on lysine 11, 48 and 63. The elongation
reaction is catalyzed by APC/C along with a different E2, UBC4
(also known as UBE2D2). These chains are recognized by the
proteasomal ubiquitin receptors, but unlike the forked chains, they
target the tagged proteins for degradation (Kirkpatrick et al., 2006).
Taken together, it appears that heterogeneous chains, in which
single ubiquitin moieties are linked to one another through different
internal lysine residues, are more common than previously thought.
Although the physiological significance of this heterogeneity is
still elusive, it is clear that forked chains do not target substrates
for degradation and instead carry out non-proteolytic functions.
Heterologous chains between ubiquitin and SUMO
SUMO is another small molecule that can also be conjugated to
lysine residues of proteins. In certain cases, it forms polymeric
chains similar to those formed by ubiquitin. Protein modification
with SUMO is involved in many cellular processes, including
signal transduction, DNA repair, stress response and targeting of
proteins to their subcellular destination (reviewed by Ulrich, 2009).

A crosstalk between SUMOylation and ubiquitylation has been
unraveled recently. In one study, it was shown that the conjugates
of SUMO2 co-purify with ubiquitin conjugates, and that SUMO2
and/or SUMO3 conjugates accumulate in cells treated with the
proteasomal inhibitor MG132. These findings suggest that
SUMOylated proteins are also ubiquitylated and targeted for
proteasomal degradation (Schimmel et al., 2008). Indeed, it has
been shown that proteins that are singly or multiply
monoSUMOylated (by SUMO2 or SUMO3) were subsequently
polyubiquitylated and degraded by the proteasome, although these
experiments did not show that SUMO and ubiquitin are linked to
one another in the same chain. Finally, the existence of mixed
SUMOubiquitin chains could be demonstrated experimentally,
but their relevance for proteasomal degradation could not be
determined (Schimmel et al., 2008). A different study demonstrated
the accumulation of SUMO1-containing conjugates following
proteasome inhibition (Matafora et al., 2009). A specific example
of heterologous modification of a substrate leading to proteasomal
degradation is the promyelocytic leukemia (PML) protein, which
is initially polySUMOylated. The SUMO chains then recruit the
ubiquitin ligase RING finger protein 4 (RNF4) (Lallemand-
Breitenbach et al., 2008; Tatham et al., 2008), which elongates the
SUMO chains by adding ubiquitin moieties to them. This, in turn,
results in proteasomal degradation of PML (Tatham et al., 2008).
Journal of Cell Science
Overall, it appears that proteins that are SUMOylated for various
non-proteolytic functions have to be ubiquitylated in order to be
targeted for degradation. However, it remains unclear whether the
ubiquitylation occurs on a SUMO residue or directly on an internal
lysine residue of the substrate. Similarly, further studies are required
to elucidate whether SUMOylation affects proteasomal recognition,
and whether substrates modified by the two types of conjugates
can be degraded.
These findings unravel yet another layer in signaling for
proteasomal degradation that involves a ubiquitin-like (UBL)
modifier. The UBL might not be involved in the proteolytic process
per se but might serve to recruit a ligase that subsequently
ubiquitylates the substrate protein.
Linear ubiquitin chains
Besides forming polyubiquitin chains, which are based on isopeptide
bonds, ubiquitin can also assemble linear chains in which the
ubiquitin moieties are linked to one another head-to-tail. The chains
are generated by the linear ubiquitin chain assembly complex
(LUBAC) ubiquitin ligase, which comprises three protein subunits,
shank-associated RH domain-interacting protein (SHARPIN), longer
isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-
1 (HOIL1L or HOIL1) and HOIL1L interacting protein (HOIP, also
known as RNF31 and ZIBRA) (Gerlach et al., 2011; Ikeda et al.,
2011; Tokunaga et al., 2011). LUBAC has been shown to promote
the degradation of ubiquitinGFP in cells, by synthesizing a linear
ubiquitin chain that is attached to the initial ubiquitin fused to GFP.
This suggests a possible role for linear chains in substrate recognition
by the proteasome (Kirisako et al., 2006). The modification of the
eukaryotic replication clamp protein PCNA [a reaction which is
mediated by the AAA ATPase cell division protein 48 (CDC48),
nuclear protein localization protein 4 (NPL4) and ubiquitin fusion
degradation 1 (UFD1) complex] with linear tetraubiquitin targets the
protein for proteasomal degradation (Zhao and Ulrich, 2010). An
additional example of a linear ubiquitin chain promoting proteasomal
degradation is provided by the model bacterial protein barstar, which
is the inhibitor of the ribonuclease barnase. N-terminally tagging
Non-canonical proteasomal signals 543
barstar with a tetraubiquitin linear chain leads to its efficient
degradation by purified proteasomes (Prakash et al., 2009).
Monoubiquitylation
As mentioned above, it has been a dominant paradigm in the
ubiquitin field that the minimal ubiquitin oligomer that is required
for recognition by the proteasome is a ubiquitin chain made of four
moieties (tetraubiquitin) (Thrower et al., 2000). However, several
recent studies have demonstrated that monoubiquitylation or
multiple monoubiquitylation can be sufficient for efficiently
targeting certain substrates for proteasomal degradation. For
instance, monoubiquitylation of paired box 3 (PAX3), an important
regulator of muscle differentiation, on a specific lysine residue
(437 or 475) by the ubiquitin ligase TAF1 (Boutet et al., 2010),
targets this protein for proteasomal degradation (Boutet et al.,
2007). Similarly, syndecan 4 (SDC4), a cell adhesion receptor that
is required for cell migration, becomes monoubiquitylated in its
cytoplasmic domain in a WNT- and DSH-dependent manner and
is subsequently degraded by the proteasome (Carvallo et al., 2010).
Furthermore, proteasomal processing of the NF-B precursor p105
to the active subunit p50 requires its modification by several single
ubiquitin moieties on a cluster of lysine residues that reside in the
C-terminal half of the molecule (Kravtsova-Ivantsiv et al., 2009).
In addition, proteasomal degradation of phospholipase D (PLD)
depends on multiple monoubiquitylation events (Yin et al., 2010).
These examples illustrate the ability of the proteasome to
recognize a variety of signals, including different polyubiquitin
chains, single monoubiquitin and a cluster of monoubiquitin
moieties, which possibly provides an additional level of specificity
in targeting proteins for degradation. In addition to the variety of
ubiquitin signals and examples of substrates discussed above, it
has been reported that ubiquitin fused to the N-terminal residues
of peptides that are longer than 20 residues, targets them for rapid
proteasomal degradation independent of further ubiquitylation
(Shabek et al., 2009). This finding raises the possibility that proteins
of a certain size (i.e. above the minimal length of 20 amino acids)
can be degraded following monoubiquitylation.
In a different study it has been shown that the primary association
of the target substrate with the proteasome depends on its
ubiquitylation and is promoted by ATP binding to the 19S subunits.
Tighter binding of the ubiquitylated protein to the proteasome
requires a loose domain in this protein and is accompanied by
hydrolysis of ATP (Peth et al., 2010). One can speculate that the
extent of ubiquitylation is increased gradually with the size of the
target substrate. The increase might be necessary in order to
generate a high enough affinity between the proteasome and the
ubiquitylated substrate to ensure its efficient and processive
degradation: a low degree of ubiquitylation on a large substrate can
destabilize its association with the proteasome, thereby rendering
the proteolytic process inefficient. This affinity hypothesis could
explain, at least in part, the diversity of the proteasomal signals and
would suggest that the proteasome has the highest affinity for a
ubiquitin chain and the lowest affinity for a single ubiquitin moiety.
It is also possible that multiple monoubiquitin moieties that interact
simultaneously with several proteasomal recognition sites increase
the affinity of the substrate to the proteasome.
Ubiquitylation of substrates on non-lysine sites
Internal sites of ubiquitylation
In some cases, protein residues other than lysine can act as ubiquitin
acceptors. For instance, cysteine, serine and threonine residues can
 544 Journal of Cell Science 125 (3)
be modified by ubiquitin, although this requires the formation of
different chemical bonds between ubiquitin and these residues than
ubiquitylation of lysine.
The BH3 interacting-domain death agonist (BID) is a member
of the B-cell lymphoma 2 (BCL2) family of antiapoptotic proteins
that has to be cleaved in order to become active. Following
cleavage, the N-terminal fragment is ubiquitylated on serine,
threonine and cysteine residues and degraded by the proteasome
(Tait et al., 2007). Another example is provided by neurogenin
(NGN), a transcription factor that has a central role in regulating
neuronal differentiation. It is ubiquitylated on canonical (lysine)
and non-canonical (cysteine, serine, threonine and the N-terminal
residue) sites, and chains with all types of internal linkages can
target the protein for proteasomal degradation (McDowell et al.,
2010; Vosper et al., 2009).
The cytoplasmic tail of the major histocompatibility complex I
(MHC I) heavy chain (HC) in the endoplasmic reticulum (ER) is
ubiquitylated on serine, threonine or lysine residues by the mouse
-herpes virus E3 ligase mK3. It is subsequently degraded by the
proteasome through the ER-associated degradation (ERAD)
pathway (Wang et al., 2007).
In this context, it is interesting to mention that cell surface MHC
class I molecules are ubiquitylated and targeted for what appears
to be lysosomal degradation following ubiquitylation on a single
Journal of Cell Science
cysteine residue (Cadwell and Coscoy, 2005). The reaction is
carried out by the MIR1 viral ligase, probably as part of the viral
strategy to inactivate the MHC class I system that would otherwise
present the viral peptides generated by the UPS to cytotoxic T cells
to ensure that the antigen-presenting cell is eliminated.
In most cases ubiquitylation occurs on lysine residues. The
ubiquitylation of non-lysine residues might reflect the ability of
cells to circumvent the structural limitations of some proteins,
whose lysine residues are not exposed, masked or lacking
altogether [see Ben-Saadon et al. for an example (Ben-Saadon et
al., 2004)]. It appears that the overall low evolutionary
conservation of ubiquitylation sites attests to the vitality and
adaptability of the UPS, which evolved, among other reasons, in
order to remove foreign, mutated and otherwise denatured and/or
misfolded proteins.
N-terminal ubiquitylation
In addition to the other non-canonical types of ubiquitylation, it
has been reported that several substrates can be ubiquitylated by
fusion of the first ubiquitin to the -NH2 group of the N-terminal
residue in a linear fashion. This linearly conjugated ubiquitin then
serves as a target for polyubiquitylation (Breitschopf et al., 1998).
Myoblast determination protein 1 (MyoD) was the first
mammalian protein that was identified as a target for N-terminal
ubiquitylation (Breitschopf et al., 1998), and a series of independent
experiments lent support to this finding. For example, mutation of
all lysine residues of MyoD only affects its degradation slightly
both in vivo and in vitro, and ubiquitylated forms of lysine-less
MyoD accumulate after inhibition of the proteasome in cells.
Furthermore, selective chemical modification of the N-terminus or
fusing of a Myc tag to the N-terminal residue, while keeping the
lysine residues intact, prevents MyoD degradation. These findings
support the notion that MyoD lacking lysine residues can
nevertheless be ubiquitylated and degraded by proteasome in an
N-terminus-dependent manner.
Examples of other proteins that are degraded through N-terminal
ubiquitylation are the Epstein-Barr virus latent membrane protein
1 (LMP1) (Aviel et al., 2000), the inhibitor of DNA binding 2
(ID2) protein (Fajerman et al., 2004), and the cyclin-dependent
kinase inhibitors p21 (also known as WAF1 and CDKN1A) (Bloom
et al., 2003; Coulombe et al., 2004), p19 (also known as ARF)
(Kuo et al., 2004) and p16INK4a (Ben-Saadon et al., 2004; Kuo et
al., 2004), the extracellular signal-regulated kinase 3 (ERK3) and
cyclin G1 (Li et al., 2009). Similarly, the peroxisome proliferator-
activated receptor co-activator 1 (PGC1) is primarily degraded
through the nuclear N-terminus-dependent ubiquitin proteasome
pathway (Trausch-Azar et al., 2010; Wang et al., 2011; Yang et al.,
2009).
In all these cases the evidence for N-terminal ubiquitylation is
largely indirect, and is based mostly on the observation that mutated
proteins lacking lysine residues can nevertheless be degraded by
the proteasome and that, in some cases, ubiquitylation still takes
place. The first direct evidence for the attachment of the C-terminal
residue of ubiquitin to the N-terminal residue of a target substrate
came from studies on the degradation of the human papillomavirus
oncoprotein-58 E7 (HPV-58 E7), where a fusion peptide
representing the two parts of the ubiquitylated protein was identified
by mass spectrometry (Ben-Saadon et al., 2004). It is possible that
N-terminal ubiquitylation, similar to ubiquitylation on residues
other than internal lysine residues, attests for the robustness of a
system that had to evolutionarily adapt to the degradation of
proteins with different structures and compositions.
Ubiquitin-independent degradation
In addition to several different types of ubiquitin modifications
that target proteins for degradation, certain proteins appear to be
degraded by the proteasome in a ubiquitin-independent manner.
For instance, it has been reported that myeloid cell leukemia 1
(MCL1) (Li et al., 2007; Stewart et al., 2010) and
CCAAT/enhancer-binding protein (C/EBP) (Zhou and Dewille,
2007) can be proteasomally degraded without prior modification
by ubiquitin.
Another example is provided by the ubiquitin-independent
proteasomal degradation of ornithine decarboxylase (ODC)
(Bercovich et al., 1989; Murakami et al., 1992). Instead of relying
on ubiquitylation, this process requires a specific chaperoning
protein, antizyme-1 (Murakami et al., 1992). Antizyme-1 binds to
the ODC monomer, which results in exposure and recognition of
the C-terminal domain of the enzyme by the 26S proteasome. It
should be mentioned, however, that ODC can be degraded by the
proteasome in the absence of antizyme-1, albeit at a substantially
reduced rate. Antizyme-1 itself is not degraded along with ODC
and is recycled: it is degraded independently in a ubiquitin-
dependent manner. Antizyme-1 has also been demonstrated to
associate and stimulate the proteasomal degradation of cyclin D1
(Newman et al., 2004) and the aurora A kinase (Lim and Gopalan,
2007a; Lim and Gopalan, 2007b). However, unlike ODC, these
proteins are also degraded in a ubiquitin-dependent manner.
Therefore, the significance and contribution of the ubiquitin-
independent pathway and antizyme-1 for their degradation is not
clear.
It has also been reported that ODC (Asher et al., 2005a), and the
tumor suppressors p53 and p73 (Asher et al., 2005b) can be
degraded by the 20S proteasome. This process is regulated by
NADH quinone oxidoreductase (NQO1), which stabilizes these
proteins: once NQO1 is inhibited by its inhibitor dicuomarol or its
expression is silenced, ODC, p53 and p73 are destabilized. The
mechanism of action of NQO1 is still not clear, but its function

Table 1. Ubiquitin ligases (E3) that synthesize non-canonical ubiquitin chains that subsequently target substrates for
proteasomal degradation
E3 ligase (E2 enzyme) Type of ubiquitin chain
CHIP Chains based on lysine 6, 11, 48 and 63
MuRF1 (UBCH1 or heterodimer Chains based on lysine 48 or 63
UBCH13UEV1)
RSP5 Lysine-63-based chain
APC/C (UBCH10, UBE2S) Lysine-11-based chain
APC/C multiple monoubiquitylation, followed by
formation of mixed chains
SIAH1 (UBCH5a) Lysine-11-based chain
RNF4 Heterologous SUMO-ubiquitin chains
LUBAC Linear
CDC48NPL4UFD1 Linear tetraubiquitin
TAF1 Monoubiquitin
mK3 Ubiquitylation on serine, threonine or lysine
residues
appears to be mediated by its binding to the substrate. As NQO1
generates NAD+ from NADH, it is possible that the mechanism of
NQO1-dependent protein stabilization is linked to either the redox
state of the cell and/or to the availability of NAD+ for ADP
Journal of Cell Science
ribosylation. It should be noted that these proteins are degraded by
the 20S complex in a ubiquitin-independent process. Because all
these proteins are also degraded by the 26S proteasome in highly
regulated processes, degradation through the NQO1-dependent
pathway must occur under unique and still to be determined
conditions. Another protein that has been reported to be degraded
by the 20S proteasome without prior ubiquitylation is BIM-extra
long [BIM(EL)], an intrinsically disordered protein that is a member
of the BCL2 family (Wiggins et al., 2011). Inhibitor of light
chain gene enhancer in B cells alpha (IB) has also been shown
to be degraded by the core 20S proteasome in a ubiquitin-
independent manner, and its degradation could be protected by the
expression of the p65 subunit of NF-B (Alvarez-Castelao and
Castano, 2005; Kroll et al., 1997). Again, IB is also degraded in
a signal- and phosphorylation-dependent manner (Alkalay et al.,
1995; Yaron et al., 1997) by the UPS. Thus the 20S- and ubiquitin-
independent pathway must be active, if at all, under unique, still to
be studied, conditions.
Taken together, these studies provide evidence that the 20S
proteasome can degrade proteins in a ubiquitin-independent manner.
However, the role the 20S proteasome has in cellular proteolysis
if it has any is highly controversial. For example, it is difficult
to explain specific substrate recognition by the 20S proteasome.
Furthermore, the 20S proteasome is inactive proteolytically, as the
N-terminal domains of the -rings interlace with one another,
thereby blocking entry of substrates into the proteolytic chamber.
Supporting this notion is the finding that the 26S proteasome
dissociates and releases free 20S complexes during the stationary
phase in S. cerevisiae, but at the same time, proteolysis rates
decrease, probably in order to protect the cell from self-digestion
during starvation. Moreover, degradation rates increase
dramatically, when the -subunits of the 20S proteasome are
mutated so that their N-terminal domains cannot interlace and thus
form a proteasome with a permanently opened gate (Bajorek et al.,
2003). It should be noted that most of the experiments suggesting
independent 20S proteasomal activity used oxidatively damaged
proteins and were carried out in cell-free systems (e.g. Davies and
Non-canonical proteasomal signals 545
Substrates References
HSP70 and HSP90 (Kundrat and Regan, 2010)
Troponin I (Kim et al., 2007)
Mga2 (Saeki et al., 2009)
BARD1, HMMR, HURP, (Song and Rape, 2010)
NUSAP1
Cyclin B1 (Kirkpatrick et al., 2006)

-catenin (Dimitrova et al., 2010)
PolySUMOulated PML (Lallemand-Breitenbach et al., 2008;
Tatham et al., 2008)
UbiquitinGFP (Kirisako et al., 2006)
PCNA (Zhao and Ulrich, 2010)
PAX3 (Boutet et al., 2010)
MHC I heavy chain (HC) (Wang et al., 2007)
Goldberg, 1987; Fagan et al., 1986; Grune et al., 2003). There is
no convincing experimental evidence to support the notion that the
20S proteasome does indeed have a role in the degradation of
proteins, even damaged ones, in cells. By contrast, there are ample
lines of experimental evidence that suggest that the degradation of
damaged proteins is dependent on the UPS. For instance, it has
been shown that the degradation of cellular proteins damaged by
heat, cadmium or paraquat requires the E2s UBC4 and UBC5, the
CDC48UFD1NPL4 ligase complex and the proteasome
(Medicherla and Goldberg, 2008). In addition, because degradation
of all types of proteins native as well as denatured requires
metabolic energy in vivo, it seems that the process must be mediated
by an intact 26S proteasome complex, the assembly and
maintenance of which are dependent on ATP. In addition,
ubiquitylation also requires energy, whereas degradation by the
20S complex is independent of energy (reviewed by Weissman
et al., 2011).
The cell cycle inhibitor p21 is degraded through two pathways
(reviewed by Lu and Hunter, 2010). Its cell-cycle-regulated
degradation is ubiquitin-dependent (Abbas et al., 2008; Amador et
al., 2007; Bornstein et al., 2003; Kim et al., 2008; Shibata et al.,
2011; Wang et al., 2005), whereas its degradation during resting
conditions is ubiquitin-independent (Chen et al., 2007; Chen et al.,
2004; Jin et al., 2003; Sheaff et al., 2000; Zhang et al., 2004).
Likewise, a dual proteasomal pathway eliminates the hepatitis C
virus (HCV) core protein. The first one is ubiquitin-dependent
(Suzuki et al., 2009) and the other is ubiquitin-independent (Yuksek
et al., 2009) but requires the proteasome activator PA28 (Suzuki
et al., 2009). However, the conclusion that the second pathway
does not require ubiquitin is based on the utilization of a mutant
form of the viral protein that does not contain lysine residues.
Therefore, the possibility that other degradation mechanisms, such
as ubiquitylation on non-lysine residues within the molecule, cannot
be excluded.
Taking into consideration these different examples, we believe
that the single well-established case of proteasome-dependent, yet
ubiquitin-independent, degradation is that of ODC, and that it is
possible there is cell-cycle-independent degradation of p21. Further
studies are required to substantiate other modes of ubiquitin-
independent degradation, in particular degradation mechanisms
that are dependent on the 20S proteasome.
 546 Journal of Cell Science 125 (3)
Conclusions and perspectives
Whereas non-canonical ubiquitin chains appear to serve diverse
non-proteolytic functions, among them activation of the NF-B
signaling pathway, regulation of DNA damage response pathways,
activation of transcriptional repressive complexes and endocytosis
(reviewed by Ikeda and Dikic, 2008), little is known about their
role in targeting proteins for proteasomal degradation.
The countless substrates of the ubiquitin system, and the
processes regulated by it, probably resulted in the evolution of a
broad repertoire of signals that can be recognized by different
ubiquitin binding domain (UBD)-containing proteins, including
shuttle proteins and the proteasome. As for ubiquitin, the complexity
and diversity of its code depend on different conformations of the
chains, which are dependent in turn on the particular lysine residue
involved in the internal linkages, the chain length and/or different
ubiquitin acceptor sites within the protein substrate. In addition,
the code might depend on other small molecule modifiers such as
ubiquitin-like proteins that can also be part of the chain.
One important, yet unsolved, question relates to the diversity of
the internal linkages. If chains made of ubiquitin moieties that are
linked through all internal lysine residues can target proteins for
degradation, why is this diversity needed? It is possible that the
different structures of the individual chains determines the strength
of their interaction with the proteasome and/or shuttle proteins, as
Journal of Cell Science
well as the recognition sensitivity of different DUBs, which might
in turn affect degradation priorities and rates among different
substrates. For example, the closed conformation of lysine-48-
based chains is clearly different from the extended conformation
of lysine-63-based and linear chains, a difference that, as noted,
might govern specific degradation characteristics. The structure of
other homogeneous chains, not to mention mixed and forked
chains, has not been resolved but can be partially predicted using
molecular modeling (Fushman and Walker, 2010). The compact
structures of lysine-11- (Bremm et al., 2010) and lysine-6-based
(Virdee et al., 2010) diubiquitin moieties has been determined and
suggests that these have a completely different structure and/or
protein surface, and hence different characteristics, than lysine-48-
and lysine-63-based chains. Clearly, additional structural analyses
and isolation and characterization of the interactomes of the
different chains are required to take the first steps towards
understanding the meaning of this diversity. It is also possible that
the functional redundancy among the various chains provides the
system with the robustness it needs to recognize the countless
protein substrates targeted by the system, including an infinite
number of conformations of even a single misfolded protein.
With regards to the E2s and E3s involved in the formation of
these chains (Table 1), studies carried out using cell-free assays
have shown that some enzymes can synthesize chains containing
all linkages, whereas others are specific, catalyzing the formation
of homogeneous chains (Kim et al., 2007; Kirkpatrick et al.,
2006). However, most of these analyses were carried out using
recombinant purified proteins in vitro, and it is not clear whether
they faithfully represent cellular events. Compared with
experimental systems, the cell contains all the E2s and E3s of the
UPS, and these can act sequentially to generate all types of
chains without one enzyme having to synthesize them all.
Importantly, and as noted above, it is possible that in the cell the
different linkages are generated spuriously rather than
intentionally, and that they do not have a physiological role in
targeting the respective substrates for degradation. Instead, it is
possible that proteins could be targeted to the proteasome by only
a short stretch of a lysine-48-based segment that is present
somewhere within the chain.
The detailed analysis of the structures of the individual chains,
and the identification of the specific proteins interacting with
different chains and their role in targeting the different substrates
for degradation, could pave the road towards the development of
specific modulators that can regulate specific substrates and
therefore processes. Thus, the inhibition of DUBs that only process
specific linkages could accelerate the degradation of substrates
tagged with such chains (by ensuring that the substrates remain
modified with long chains that are recognized more efficiently by
the proteasome), such as, for example, aggregated proteins [see
Lee et al. for an example (Lee et al., 2010)]. The inhibition of a
different subset of DUBs, however, could inhibit the proteasome
by not allowing removal of the chain, and thereby keeping the
tagged substrates bound to the proteasome and blocking the access
for other ubiquitylated substrates (e.g. DArcy et al., 2011).
An additional unsolved question that needs to be addressed in
future studies is the identity of proteasomal subunits and shuttle
proteins. Because these proteins are responsible for the recognition
of the numerous ubiquitin chains, and possibly the recognition of
multiple single ubiquitin moieties, their identification and
characterization will provide additional insight into the regulation
of this complex system.
Funding
Research in the laboratory of A.C. is supported by grants from the Dr
Miriam and Sheldon Adelson Foundation for Medical Research
(AMRF); the Israel Science Foundation (ISF); the German-Israeli
Foundation for Research and Scientific Development (GIF); and the
Deutsch-Israelische Projektkooperation (DIP). A.C. is an Israel Cancer
Research Fund (ICRF) USA Professor.
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